The long term goal of this research is to build a model of the engines that mediate co- and post-translational translocation at the endoplasmic reticulum (ER). Hence, this work will provide insights into the structure and function of the ribosome-Sec61p and SEC complexes, respectively. We will use 3D electron cryo-microscopy to determine structures of the mammalian, ribosome-channel complex (RCC) and its associated membrane proteins. This ensemble includes TRAP, OST and TRAM. The 3D structure of the yeast SEC complex will also be investigated. Together, these translocons mediate essential steps in the biogenesis of membrane proteins, secretory proteins and resident ER proteins. We have shown that the Sec61p complex forms an asymmetric channel with 3-4 subunits, in both the co- and post-translational pathways. In a stepwise approach, we have solved 3D structures of the native RCC in a ground state at resolutions of approximately 27 and 17A. We are now extending the 3D structure of the RCC to 11A resolution and will push this specimen to approximately 8A resolution, to reveal the (-helical nature of the channel. This will provide unique insights into channel assembly, gating and protein translocation. In addition, the structure of the attendant mammalian ribosome will allow direct comparisons with models of prokaryotic and yeast ribosomes. In parallel studies, we will identify the membrane protein that associates with Sec61p to form a large, lumenal domain on the native channel. Single particle studies will also be carried out on OST and on larger "STO" complexes which contain Sec61p, TRAP and OST, to better understand the interactions of these components within the translocon. To complement this work, attempts will be made to grow 3D crystals of the lumenal domain of TRAP which is likely comprised of an (( heterodimer. Finally, a nearly complete translocon that includes Sec61p, TRAP, OST, TRAM, SR and the ribosome has been isolated. A structure determination by EM of this complex may reveal the relative orientations of these membrane proteins and provide insights into their functions during translocation.